Osmium compounds

ABSTRACT

This specification describes a complex of osmium tetroxide and a compound having a heterocyclic ring containing one or two tertiary nitrogen atoms. The compound may be an aromatic tertiary amine and the tertiary nitrogen atom may be the sole heterocyclic atom in the heterocyclic ring. The complexes described are suitable for fixing and staining cells and may be used in the preparation of biological specimens for examination by electron microscopy.

This is a division of application Ser. No. 702,132 filed July 2, 1976,now U.S. Pat. No. 4,100,158.

This invention relates to osmium tetroxide complexes, and in particularto complexes suitable for fixing and staining cells, particularly in thepreparation of biological specimens for examination by electronmicroscopy.

The technique of electron microscopy has, in recent years, enabledstartling advances in the field of biological research to be made. Theprincipal advantage of the electron microscope is that it can be used toexamine objects up to at least a thousand times smaller than a lightmicroscope can resolve, but a disadvantage is that, unlike the lightmicroscope, the electron microscope cannot examine living cells.Biological specimens therefore have to be prepared prior to examination.

One way of preparing specimens is the "histological" method, the aim ofwhich is to preserve the specimen faithfully and exactly as it was inlife and to render clearly visible all the inter-relationships betweentissues, cells, organelles and macro-molecules. This is achieved byadding to the living cells a substance which will kill the tissues and,at the same time, preserve all the fine structure and prevent anypost-mortem reactions or attack by micro-organisms from taking place.This process is known as "fixation". However, the penetrating power ofan electron beam is not sufficiently great to traverse a section of thethickness of even a single cell, and so cells must be sliced intosections of 500 A thick or even thinner.

Having regard to the above constraints, the stages in electron histologyare basically as follows:

(I) Fixation: killing the specimen with preservation of the finestructure;

(II) Block staining: causing certain components of the structure toattract heavy metal ions and thus to scatter electrons differentially inorder to render the structure visible in the electron microscope.(Stages (i) and (ii) may be combined);

(III) AND (IV) Dehydration and Infiltration: removing water andreplacing it with a fluid which can be hardened by, for example,polymerisation into a solid which is sufficiently elastic to be slicedevenly into sections of thickness 500A or less;

(V) Polymerisation;

(VI) Sectioning;

(VII) Mounting sections on copper support grids; and

(VIII) Section staining; increasing the existing differential electronscattering power of the specimen constituents by renoting with solutionsof heavy metals.

The primary purpose of the fixative--that is, the substance used to"fix" the specimen--is to solidify the protein sol which flows in thenetwork of phospholipoprotein membranes forming the cell framework. Itshould also render insoluble all the other chemical constituents of thecell, such as nucleic acids, nucleo-proteins, carbohydrates and lipids.The fixative should also provide electron contrast, thus making itpossible to combine stages (i) and (ii) in the above-mentioned scheme.

However, it is impossible in practice to satisfy all these conditionswith one fixative, but the substance that has hitherto been used most,and which has come closest to satisfying all these conditions, is osmiumtetroxide. This substance stabilizes protein sols by chemicalcombination and formation of cross-linkages and preserves, almostperfectly, coll fine structure. It preserves lipids, phospholipoproteinsand nucleoproteins but is a poor preservative for carbohydrates andnucleic acids. Above all, it is an excellent producer of electroncontrast. This last property is due to the fact that, since osmiumtetroxide fixes by chemical combination rather than precipitation,osmium metal remains in the fixed cell, attached firmly to the structureit stabilizes, thus--due to its extremely high density--delineating thestructure almost perfectly in an electron beam. Furthermore, osmiumtetroxide does not harden and embrittle fixed tissue, neither does itshrink or swell the fixed tissue.

In practice, specimens are usually subjected to a two-stage fixingprocedure, the first stage of which is designed to overcome the fewdisadvantages possessed by osmium tetroxide. For example, apart from thealready-mentioned poor performance in preserving carbohydrates andnucleic acids, osmium tetroxide penetrates tissues only very slowly. Thefirst stage of the two-stage process often consists of an aldehydefixation, frequently using glutaraldehyde, and the second stage consistsof the osmium tetroxide fixation.

The remainder of the stages in the electron histological method ofspecimen preparation need only be mentioned in passing, because theyhave no direct relevance to the present invention.

The fluid for the infiltration stage is selected according to therequired properties of the resin in which the specimen is ultimately tobe embedded. A common type of resin in use today is a thermosettingsynthetic epoxy resin, for example "Araldito" (Registered Trade Mark),often with a plasticiser such as dibutyl phthalate added to make thecured resin more suitable for sectioning.

The mounted sections are optionally stained to increase further thedifferential electron scattering power initially induced by osmiumtetroxide. The section is reacted with a solution of a heavy metal salt,for example alkaline lead citrate. Lead is especially useful forstaining glycogen, which is unstained by osmium tetroxide. Other stainswhich may be used for specific purposes are, for example, aqueous oralcoholic uranyl acetate, aqueous or alcoholic phosphotungstic acid andaqueous potassium permanganate.

It is an object of the present invention to provide a more convenientsource of osmium for fixing and/or staining cells. Osmium tetroxide isvery volatile and the vapour thereof is extremely toxic and,consequently, considerable care must be exercised in its storage,handling and use. It should always be used, for example, in an efficientfume cupboard, with the front pulled down as far as possible, andhandled only with rubber gloves. Furthermore, it is usually supplied insealed vials and, once the vial is opened, it is the general practice todiscard what is not immediately required rather than store it for futureuse.

It is known that one convenient source of osmium tetroxide is the 2:1addition compound formed from osmium tetroxide andhexamethylene-tetramine, having the molecular formula C₆ H₁₂ N₄.2OsO₄.This compound is a stable crystalline solid, melting at 180°-185° C. andis non-explosive under the influence of heat. One great advantage ofthis compound is that, in the dry, solid state, the vapour pressure ofthe osmium tetroxide is extremely low. It may therefore be handledfreely on the open laboratory bench and, unlike osmium tetroxide, it maybe stored for long periods after its container seal has been broken.Like osmium tetroxide, the addition compound of hexamethylene-tetramineand osmium tetroxide is only soluble with difficulty in cold water, butsolutions may readily be prepared by first dissolving the compound inthe minimum amount of dimethylformamide and then diluting to the desiredconcentration with distilled water. The complex dissociates and thesolution has a vapour pressure of osmium tetroxide similar to aqueoussolutions of osmium tetroxide of comparable strength.

We have now found according to the present invention that complexes ofosmium tetroxide and certain heterocyclic compounds are useful for thefixing and/or staining of cells in the preparation of specimens forexamination by electron microscopy. These complexes are new compounds.

Accordingly, a first aspect of the invention provides complexes ofosmium tetroxide and a compound having a heterocyclic ring containingone or two tertiary nitrogen atoms.

Said compound can be an aromatic tertiary amine or a cycloaliphatictertiary amine. Where it is an aromatic tertiary amine, it can be aheterocyclic compound having only one tertiary nitrogen atom in theheterocyclic ring, for example, isoquinoline, or it can be aheterocyclic compound having two tertiary nitrogen atoms in theheterocyclic ring, for example, pyridazine or phthalazine.

Where said compound is a cycloaliphatic tertiary amine, it can be onehaving only one tertiary nitrogen atom in the heterocyclic ring, forexample, quinuclidine, or it can be a heterocyclic compound having twotertiary nitrogen atoms in the heterocyclic ring, for example,triethylene diamine (i.e. 1,4-diazabicyclo-2,2,2,-octane).

The complexes of the invention are believed to be straight-forwardaddition complexes and they can be prepared, in general, by adding theheterocyclic compound to an aqueous solution of osmium tetroxide andisolating the product.

Accordingly, in a second aspect, the invention provides a process forthe production of osmium tetroxide complex which comprises reactingosmium tetroxide with a compound having a hetrocyclic ring containingone or two tertiary nitrogen atoms, and recovering the resultingproduct.

The following examples illustrate in detail the preparation of somecomplexes according to the invention. The amines (heterocycliccompounds) used in these examples are:

    ______________________________________                                        Example Amine       Structure      Formula                                    ______________________________________                                        1       pyridazine                                                                                 ##STR1##      C.sub.4 H.sub.4 N.sub.2                    2       phthalazine                                                                                ##STR2##      C.sub.8 H.sub.6 N.sub.2                    3       isoquinoline                                                                               ##STR3##      C.sub.9 H.sub.7 N                          4       triethylene- diamine                                                                       ##STR4##      C.sub.6 H.sub.12 N.sub.2                   5       quinuclidine                                                                               ##STR5##      C.sub.7 H.sub.13 N                         ______________________________________                                    

example 1

osmium tetroxide (3 g) was dissolved in water (120 ml) at 18°-22° C.Pyridazine (2.2 g) was added dropwise. Yellow crystals were precipitatedwhich were filtered, washed with water and a minimum quantity of ethanoland dried in air. Yield=0.9 (23% of theoretical).

Solubility in dimethylformamide: 420 g. 1⁻¹.

    ______________________________________                                        Formula of complex: OsO.sub.4 . C.sub.4 H.sub.4 N.sub.2 .                     Elemental analysis:                                                                          C         N         O                                          ______________________________________                                        Calculated     14.36     8.38      19.15                                      Found          14.30     8.40      18.62                                      ______________________________________                                    

EXAMPLE 2

Osmium tetroxide (5 g) was dissolved in water (200 ml) at 18°-22° C.Phthalazine (2.8 g) was dissolved in water (45 ml) and added to thesolution of osmium tetroxide. The resultant yellow crystals werefiltered, washed in water and dried in air.

Yield=6.6 g (86% of theoretical)

Solubility in dimethylformamide: 550 g. 1⁻¹.

    ______________________________________                                        Formula of complex: OsO.sub.4 . C.sub.8 H.sub.6 N.sub.2                       Elemental analysis:                                                                          C         N         O                                          ______________________________________                                        Calculated     25.00     7.29      16.67                                      Found          25.58     7.52      16.75                                      ______________________________________                                    

EXAMPLE 3

Osmium tetroxide (5 g) was dissolved in water (200 ml) at 18°-22° C. andisoquinoline (2.8 g) was added dropwise with stirring over a period ofabout half an hour. The yellow crystalline product was filtered, washedwith water and a minimum volume of ethanol and air dried.

Yield=5.7 g (75% of theoretical).

Solubility in dimethylformamide: 500 g. 1⁻¹.

    ______________________________________                                        Formula of complex: OsO.sub.4 . C.sub.9 H.sub.7 N                             Elemental analysis:                                                                          C         N         O                                          ______________________________________                                        Calculated     28.18     3.65      16.70                                      Found          28.60     3.69      16.41                                      ______________________________________                                    

EXAMPLE 4

Osmium tetroxide (5 g) was dissolved in water (200 ml) at 18°-22° C. andtriethylenediamine (1.1 g) in water (20 ml) was added with stirring overfive minutes. The orange-red microcrystalline product was filtered,washed with water and air dried.

Yield=5.4 g (87% of theoretical).

Solubility in dimethylformamide: 200 g. 1⁻¹.

    ______________________________________                                        Formula of complex: 2 OsO.sub.4 . C.sub.6 H.sub.12 N.sub.2                    Elemental analysis:                                                                          C         N         O                                          ______________________________________                                        Calculated     11.61     4.51      20.63                                      Found          11.63     4.52      20.32                                      ______________________________________                                    

EXAMPLE 5

Osmium tetroxide (5 g) was dissolved in water (200 ml) at 18°-22° C.Quinuclidine hydrochloride (5 g) was dissolved in the minimum quantityof water with sodium hydroxide (1.36 g) present and added to the osmiumtetroxide solution with stirring for five minutes. The resultantorange-red crystals were filtered, washed with water and air dried.

Yield = 7.2 g (99% of theoretical).

Solubility in dimethylformamide: 175 g. 1⁻¹.

    ______________________________________                                        Formula of complex: OsO.sub.4 . C.sub.7 H.sub.13 N                            Elemental analysis:                                                                          C         N         O                                          ______________________________________                                        Calculated     23.00     3.83       17.52                                     Found          23.03     3,84      17.34                                      ______________________________________                                    

The infra-red spectra of all the complexes showed characteristicabsorptions for osmium-oxygen multiple bonds in the 890-950 cm⁻¹ region.All spectra also showed absorptions in the 300-400 cm⁻¹ region, probablydue to osmium-oxygen bonding.

The complexes according to the invention provide more convenient sourcesof osmium tetroxide than is afforded by the use of osmium tetroxideitself. The complexes all possess the favourable properties alsopossessed by the complex formed from osmium tetroxide andhexamethylenetetramine, that is, stability and low vapour pressure ofosmium tetroxide at ordinary temperatues.

Complexes of this invention are soluble in dimethyl formamide and can beused, for example, as a solution in that solvent (preferably in amixture of that solvent with water) in the fixing and/or staining ofcells.

In a third aspect, the present invention provides a method of fixing orstaining cells in the preparation of specimens for examination byelectron microscopy, in which the cells are treated with a complex ofthis invention.

Solutions of complexes according to the invention may be used in thesame way as solutions of osmium tetroxide, that is, they generallyprovide secondary fixing and staining solutions following a primary fixin glutaraldehyde, for example.

Complexes according to the invention may also be used as replacementsfor osmium tetroxide in other applications. For example, they may beused to form osmium blacks in electron microscopic cytochemistry.Further, they may be used as replacements for osmium tetroxide in thecis-hydroxylation of olefins. The general procedures for the formationof osmium blacks are described in Data Sheet 180-8/74 by OlysciencesInc., entitled "Osmeth" and the general procedures for thecis-hydroxylation of olefins are described in Organic Chemistry, by I.L. Finar, published by Longmans, Vol 2, 2nd Edition, pages 113, 114,117.

What we claim is:
 1. In a method of fixing or staining cells in thepreparation of specimens for examination by electron microscopy, inwhich the cells are treated with osmium tetroxide, the improvementcomprising employing for said osmium tetroxide a complex of osmiumtetroxide and a compound having a heterocylic ring containing one or twotertiary nitrogen atoms said compound being selected from the groupconsisting of pyridazine, phthalazine, isoquinoline, triethylenediamine,and quinuclidine.
 2. A method according to claim 1, in which the complexis in solution in aqueous dimethyl formamide.
 3. In a method of formingosmium blacks in electron microscopic cytochemistry, which comprisestreating cells with osmium tetroxide, the improvement comprisingemploying for said osmium tetroxide a complex of osmium tetroxide and acompound having a heterocyclic ring containing one or two tertiarynitrogen atoms said compound being selected from the group consisting ofpyridazine, phthalazine, isoquinoline, triethylenediamine, andquinuclidine.